The NIMH transgenic core facility has several major functions: 1) to produce transgenic research animals for neuroscience research, 2) to support research with associated techniques in genetic research in neuroscience, 3) to develop new transgenic techniques and model systems and 4) to engage in collaborative projects that promote genetic approaches to neuroscience research. 1) Production Metrics of production over the past year include: a) 19 transgenic mouse projects produced by oocyte injection of DNA or CRISPR constructs, with multiple lines produced for each project. b) Five projects using embryonic stem cells (ESC) have been undertaken. c) Three transgenic rat projects produced by oocyte injection, with multiple lines produced for each project. 2) Technical Support a) 87 transgenic rodent lines have been archived by cryopreserving germ cells or embryos. b) 39 lines have been re-derived, by transferring lines from pathogen-bearing animals into those with defined health status. c) Transgenic project design and assistance have continued to be significant to NIH neuroscience labs that lack experience in producing transgenic animals. 3) Technical development a) Nuclease-mediated genetic engineering (CRISPR): Mouse embryonic development has been observed and manipulated for decades. Genetic manipulation has taken advantage of the ability to grow totipotent cells (embryonic stem cells, ESC) in a dish. Other species, including primates, have been refractory to the same technique that works so well for mice. The result has been an ever-expanding collection of transgenic mouse lines that scientists use to test the role of virtually every one of the roughly 20,000 genes that make up a mouse genome. Now, the CRISPR/Cas9 system can be used to target genes more efficiently and as specifically as in mouse ESC, but is applicable to other species. The core has focused on using CRISPR/Cas9 to generate conditional knock out genes in mice, rats and primates. In these animals, recombination signals are inserted to flank critical regions of a gene. Only when a recombinase is expressed in the cell is the gene function disrupted. Subtle changes in the gene can also be made, but making a conditional modification replaces large segments of a gene. The core has over the last year benefited from a collaboration with Nick Ryba in the National Institute of Dental and Craniofacial Research (NIDCR) to improve the efficiency of modifying genes by replacing large segments. We have optimized concentration of the injected material, the use of reagents to optimize homologous recombination of donor sequences, the site of injection in embryos, and the construction of nucleic acid targeting vectors. These techniques are now being applied to make mouse and rat transgenics, with the ultimate aim of making the technique efficient enough to use in species with smaller numbers of offspring, and longer gestation and maturation times, such as marmosets. b) Transgenic marmosets: In collaboration with Erika Sasaki at the Central Institute for Experimental Animals in Kawasaki, Japan the core has produced a line of transgenic marmosets that expresses a genetically encoded calcium indicator (GECI) which will allow the visualization of neuronal activity in vivo. To expand a line of animals that express this gene, we have developed an artificial insemination procedure that has resulted in multiple pregnancies in the NIMH colony and in the Xiaoqin Wang's colony at Johns Hopkins University. These animals are being used for experiments on the activity in the sensory cortex. c) Transgenic rat lines: Over 50 laboratories from around the world have requested transgenic rat lines that were produced by the transgenic core in collaboration with labs in NIDA. These lines are being maintained and distributed by the RRRC (Rat Resource and Research Center). More than ten of these lines that express CRE recombinase in specific neuronal subtypes are continuing to be characterized for their patterns of expression. An inbred line of rats that expresses the orange fluorescent protein (OFP) is being used by Alan Korestsky's group (NINDS) to study the potential of transplanted neural stem cells. d) Support techniques: several techniques are under development to increase the capacity of the core's support functions. Freezing mouse sperm and improving IVF by using newer methods is a major effort. Freezing rat sperm and achieving IVF in rats at an acceptable level is a challenging task in all laboratories. A method of introducing adeno-associated virus (AAV) to the brain through the peripheral circulation is also underway. This method would allow more widespread delivery of a transgene and eliminate the need for intracranial delivery by surgical intervention.